Download Protein Folding using Fluorescence Spectroscopy

Imperial Journal of Interdisciplinary Research (IJIR)
Vol-3, Issue-2, 2017
ISSN: 2454-1362, http://www.onlinejournal.in
Protein Folding using Fluorescence Spectroscopy
Divya , Walia Neha & Yadav Priyanka
1,2
Student, 3Assistant professor
1,2,3
FLTMSBP Govt. College of women , Rewari ( hr.) , India , department of chemistry
Abstract: Proteins are the large macromolecules,
which consists of one or more long amino
acid residues. They differ from one another
primarily in their sequence of amino acids.The
sequence of Amino Acids is dictated by
the nucleotide sequence of results in folding of the
protein into a specific three-dimensional structure .
In proteins, the three aromatic amino acids—
phenylalanine, tyrosine, and tryptophan—are all
fluorescent. These three amino acids are relatively
rare in proteins. If all twenty amino acids were
fluorescent then protein emission would be more
complex.
Protein Folding
Protein folding is the process by which
a protein structure assumes its functional shape or
conformation. It is the physical process by which
a polypeptide folds into its characteristic and
functional three-dimensional structure from random
coil.
Process of Protein Folding
The process of folding begins so that the Nterminus of the protein begins to fold while the Cterminal portion of the protein is still being
synthesized .-chains exposed to water is an
important driving force behind the folding
process16. Formation of intramolecular hydrogen
bonds provides another important contribution to
protein stability. The strength of hydrogen bonds
depends on their environment.17Many proteins take
at least a few seconds to fold.
Figure 13: PROTEINS BEFORE FOLDING AND
AFTER FOLDING
Fluorescence Spectroscopy : A Tool For
Studying Protein Folding
When a molecule or an atom absorbs light it can go
into the excited stage. Due to absorption of light
molecule in the excited stage can re-emit the
absorbed light and come back to the ground stage.
The excited molecule can go into the two possible
excited states: singlet and triplet. Molecule in the
singlet state when returns to the ground state emit
light: the process is called fluorescence and the
spectrum of the emitted light is called Fluorescence
Spectroscopy. Molecule in the triplet excited state
also emits light when returns the ground state: this
process is called Phosphorescence. Fluorescence
spectroscopy can be understood easily by Jablonski
Diagram. The processes that occur between the
absorption and emission of light are usually
illustrated by the Jablonski diagram. These
diagrams are named after Professor Alexander
Jablonski who is regarded as the father of
fluorescence spectroscopy .
Figure 15: jablonskii diagram
Imperial Journal of Interdisciplinary Research (IJIR)
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Imperial Journal of Interdisciplinary Research (IJIR)
Vol-3, Issue-2, 2017
ISSN: 2454-1362, http://www.onlinejournal.in
The singlet ground, first, and second electronic
states are depicted by S0, S1, and S2 respectively.
At each of these electronic energy levels the
fluorophores can exist in a number of vibrational
energy levels, depicted by 0, 1, 2, etc. The
transitions between states are depicted as vertical
lines to illustrate the instantaneous nature of light
absorption. Transitions occur in about 10–15 s, a
time too short for significant displacement of
nuclei. This is the Franck-Condon principle.
At room temperature thermal energy is not
adequate to significantly populate the excited
vibrational states. Absorption and emission occur
mostly from molecules with lowest vibrational
energy. The larger energy difference between the
S0 and S1 excited states is too large for thermal
population of S1. For this reason we use light and
not heat to induce fluorescence.
A fluorophore is usually excited to some higher
vibrational level of either S1 or S2. This process is
called internal conversion and generally occurs
within 10^ (-12) s or less. Since fluorescence
lifetimes are typically near 10^ (-8) s, internal
conversion is generally complete prior to emission.
Hence ,fluorescence emission results from a
thermally equilibrated excited state, the lowest
energy vibrational state of S1.Return to the ground
state typically occurs to a higher excited vibrational
ground state level, which then quickly(10–12
s)reaches thermal equilibrium. An interesting
consequence of emission to higher vibrational
ground states is that the emission spectrum is
typically a mirror image of the absorption spectrum
of the S0 ―› S1 transition.
Molecules in the S1 state can also undergo a spin
conversion to the first triplet state T1. Emission
from T1 is termed phosphorescence, and is
generally shifted to longer wavelengths relative to
the fluorescence. Conversion of S1 to T1 is called
intersystem crossing. Transition from T1 to the
singlet ground state is forbidden.. Molecules
containing heavy atoms such as bromine and iodine
are frequently phosphorescent. The heavy atoms
facilitate intersystem crossing and thus enhance
phosphorescence quantum yields.
exceeds about 0.05 in a 1 cm path length, the
relationship
becomes
nonlinear.
Because
fluorescence quantization is dependent on the
instrument, fluorescent reference standards are
essential for calibrating measurements made at
different times .
Background Fluorescence :
Fluorescence detection sensitivity is severely
compromised by background signals, which may
originate from endogenous sample constituents
(referred to as auto fluorescence) or from unbound
or non-specifically bound probes (referred to as
reagent
background).
Detection
of
auto
fluorescence can be minimized either by selecting
filters that reduce the transmission Furthermore, at
longer wavelengths, light scattering by dense media
such as tissues is much reduced, resulting in greater
penetration of the excitation light.
Fluorescent Proteins
Proteins contain three amino-acid residues that
contribute to their ultraviolet fluorescence. These
are tyrosine (tyr, Y), tryptophan (trp, W), and
phenylalanine (phe, F).Tyrosine contains benzene
ring and hydrophilic –OH group while tryptophan
has indole ring attached through a methylene
group. Due to delocalization of the π- electrons in
these two, these are highly fluorescent.
The absorption and emission spectra of these amino
acids are shown in Figure;
Fluorescence Signals :
Fluorescence intensity is quantitatively dependent
on the same parameters as absorbance defined by
the Beer–Lambert law as the product of the molar
extinction coefficient, optical pathlength and solute
concentration — as well as on the fluorescence
quantum yield andthe excitation source intensity
and fluorescence collection efficiency of the
instrument. In dilute solutions or suspensions,
fluorescence intensity is linearly proportional to
these parameters. When sample absorbance
Imperial Journal of Interdisciplinary Research (IJIR)
Figure 16: Absorption and emission spectra of aromatic
amino acids33
Emission of proteins is dominated by tryptophan,
which absorbs at the longest wavelength Energy
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Imperial Journal of Interdisciplinary Research (IJIR)
Vol-3, Issue-2, 2017
ISSN: 2454-1362, http://www.onlinejournal.in
absorbed by phenylalanine and tyrosine is often
transferred to the tryptophan residues in the same
protein.
Phenylalanine displays the shortest absorption and
emission wavelengths. Phenylalanine displays a
structured emission with a maximum near 282 nm.
The emission of tyrosine in water occurs at 303 nm
and is relatively insensitive to solvent polarity. The
emission maximum of tryptophan in water occurs
near 350 nm and is highly dependent upon polarity
and/or local environment. Indole is sensitive to
both general solvent effects. Indole displays a
substantial spectral shift upon forming hydrogen
bond to the amino nitrogen, which is a specific
solvent effect. Additionally, indole can be
quenched by several amino-acid side chains.
Consequently, phenylalanine is not excited in most
experiments. Furthermore, the quantum yield of
phenylalanine in proteins is small—typically near
0.03—so emission from this residue is rarely
observed.
The absorption of proteins at 280 nm is due to both
tyrosine and tryptophan residues. At wavelengths
longer than 295 nm, the absorption is due primarily
to tryptophan. Tryptophan fluorescence can be
selectively excited at 295–305 nm. Tyrosine is
often regarded as a rather simple fluorophore..
Tyrosine can undergo excited-state ionization,
resulting in the loss of the proton on the aromatic
hydroxyl group.34
Imperial Journal of Interdisciplinary Research (IJIR)
Phenylalanine absorbs light in the visible region.
TRYPTOPHAN/TYROSINE
FLUOROSCENCE
There are three amino acids with intrinsic
fluorescence properties, phenylalanine (Phe), and
tyrosine(Tyr) and tryptophan (Trp) but only
Tyrosine and tryptophan are used experimentally
because their quantum yields is high enough to
give a good
Fluorescence signal.
For an excitation wavelength of 280 nm, both Trp
and Tyr will be excited. To selectively excite
Trp only, 295 nm wavelength must be used.
Those residues can be used to follow protein
folding because their fluorescence properties
(Quantum yields) are sensitive to their environment
which changes when
protein folds/unfolds. In the native folded state, trp
and try are generally located within the core of the
protein, whereas in a partially folded or unfolded
state, they become exposed to solvent .In a
hydrophobic environment (buried within the core
of the protein), Tyr and Trp have a high quantum
yield and therefore high fluorescence intensity. In
contrast in a hydrophilic environment (exposed to
solvent) their quantum yield decreases leading to
low fluorescence intensity. For Trp residue, there is
strong stoke shifts dependent on the solvent,
meaning that the maximum emission wavelength of
Trp will differ depending on the Trp environment.
There are several means to unfold a protein based
on the disturbance of the weak interactions that
maintains the protein folded (hydrogen bonding,
electrostatic
interactions,
and
hydrophobic
interactions).
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Imperial Journal of Interdisciplinary Research (IJIR)
Vol-3, Issue-2, 2017
ISSN: 2454-1362, http://www.onlinejournal.in
Figure 17: PROTEIN FOLDING AS OBSERVED IN TRYPTOPHAN FLUORESCENCE.37
The most common ways of unfolding a protein are
chaotropic agents (urea, guanidium hydrochloride),
the change of pH (acid, base) or the rise of
temperature. It is possible to study either steady
state or kinetic of protein unfolding. For example,
the protein is unfolded by increasing temperature,
so at each temperature the protein undergo
unfolding and reach an
Equilibrium state corresponds to a partially folded
or fully unfolded state depending on the conditions.
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